Primary CNS GCT consists of several subtypes with different degrees of histological differentiation and malignancy. According to histological differentiation, related tumor markers, and secreted protein markers, these tumors can be classified into germinomas and nongerminomatous GCTs (NGGCTs), the latter including embryonal carcinoma (EC), yolk sac tumors (YST), choriocarcinoma (CC), teratoma (mature teratoma, immature teratoma, or immature teratoma with malignant differentiation) and mixed GCTs [6]. For NGGCTs, except for benign mature teratoma, all of the other tumors present with diverse malignancies and therapeutic sensitivities when compared to germinomas and are grouped together as nongerminomatous malignant GCTs (NGMGCTs). NGMGCTs require more extensive drug and irradiation treatment regimens, have a higher recurrence rate and a lower survival rate [7,8]. Clinically, >50% of pediatric CNS GCTs are germinomas, while the majority of remaining tumors are NGMGCTs [5,9]. Histologically, germinoma is the most undifferentiated GCT and is composed of undifferentiated large cells that resemble primordial germinal elements. Among the NGGCTs, the histological picture differs depending on the diagnosis. EC contains undifferentiated stem cells resembling the embryonic inner cell mass (ICM). YST and CC correspond to the extra-embryonic differentiation along mesoblast and trophoblast lines, respectively. This contrasts with teratomas, which consist of differentiated derivatives that include all three germ layers with or without incompletely differentiated tissue elements, like neuroepithelium, which resembles fetal tissue. CNS GCTs often present with more than one histological component and are then classified as mixed GCTs [7,10,11].

GCTs are presumed to arise from mutated primordial germ cells (PGCs) of genital ridge origin or dysfunction totipotent embryonic cells [12]. Investigation of the different genetic compositions in ECs and ES cells may provide clues about the reduced dependency on external cues for self-maintenance that exist among GCTs, thereby benefiting tumorigenesis research on ECs as well as applications for human ES cells (see also a review article by Werbowetski-Ogilvie et al. [13]). Global gene expression studies in human embryonic stem cells and human pluripotent germ cell tumors have shown that the gene expression patterns of human ES cell lines are similar to those of the human embryonal carcinoma cell samples but are more distantly related to those of seminoma samples [12,14]. Genes that are expressed at significantly greater levels in human ES and embryonal carcinoma cell lines than in control samples were pinpointed and are possible candidates for involvement in the maintenance of a pluripotent undifferentiated phenotype [12]. Wnt and Notch pathway genes are overexpressed in the pluripotent human embryonal carcinoma cell line NTERA2 and in embryonic stem cells [15]. These include members of the frizzled gene family (FZDI, FZD3, FZD4, FZD5, FZD6), which encodes receptors for the Wnt proteins, the Frizzled Related Protein family (SFRPI, SFRP2, FRZB, SFRP4), which encode soluble Wnt antagonists and also ligands and receptors of the Notch pathway (Dlkl, Jaggedl; Notchl, Notch2, Notch3) [15].

The histological differences between the various different GCTs are mirrored by their gene expression profiles [16,17]. Genomic studies have been conducted on GCTs, most notably on Caucasian adult gonadal ones [12,16]. However, only limited gene profiling studies have focused on primary pediatric CNS GCTs, and, to our knowledge, no transcriptome profiling work on Asian cases has been reported. A very recently paper studied global mRNA expression patterns in pediatric malignant GCTs arising from the testis, the ovary, the sacrococcygeal region and the brain, and then compared these with adult testicular tumors. These results showed that there is no segregation of GCTs with the same histology at different sites or at different ages, within the pediatric range. However, clear segregation of pediatric and adult tumors, most conspicuously among the YSTs, was observed [17]. The pediatric seminomas are significantly enriched for genes associated with a self-renewing pluripotent phenotype, whereas the pediatric YSTs are significantly enriched for genes associated with differentiation and proliferation [17]. These results suggest that the observed clinical differences between pediatric CNS GCTs from different ethnic backgrounds or prognosis groups may also be detected using genomic analysis.

MicroRNAs (miRNAs) are small RNAs of 18-24 nucleotides in length that are involved in the regulation of gene expression and hence a variety of biological processes through post-transcriptional RNA interference-based mechanisms. Matured miRNAs interact and inhibit target mRNAs and result in translational repression or mRNA cleavage [18-20]. In medulloblastoma (MB), an aggressive brain malignancy with a predominant incidence in childhood, a high throughput miRNA profiling analysis found that only a few miRNAs displayed upregulated expression, while most of them, such as miR-9 and miR-125a, were downregulated in the tumor samples, suggesting a tumor growth-inhibitory function [21]. Moreover, the same group identified miRNAs downregulated in human MBs with high Hedgehog (Hh) signaling, which is one of the pathogenesis mechanisms of MB [22]. Differential miRNAs, such as miR-184, have been identified and found to correlate with prognosis, differentiation, and apoptosis in pediatric neuroblastoma [23]. A high-throughput miRNome analysis of adult gonadal GCTs has been published, and in each GCT subtype the miRNA patterns are quite different [24]. For GCTs in children, only limited miRNA data has been reported.

Genomic copy number variation (CNV) in GCTs of adulthood has been extensively investigated. Gain of 12p in up to 80% of cases of adult testicular GCTs [25,26]. In contrast, comparatively little genomic CNV investigation has been conducted on childhood GCTs. Using metaphase comparative genomic hybridization (CGH), a wide range of CNVs has been described in pediatric GCTs, including gains on 1q, 2p, 3, 7, 8, 13, 14, 20q, 21, and X, as well as losses on 1p36, 4q, 6q, 11, 13 and 18; but none are seen consistently [27-29]. This may due to either the heterogeneity of the GCTs, or the different algorithms that were applied to identify the CNV regions. In 2007, Palmer et al. used 34 GCTs (22 yolk sac tumors (YSTs), 11 germinomatous tumors and one metastatic embryonal carcinoma), which had occurred in children from birth to age 16, for CNV analysis. Most of their cases were from the testis, the ovary and the sacrococcygeal region and only two germinomas and one YST brain BCT were included [30]. Gain of 12p was found to be present in 53% of primary MGCTs of children aged 5-16 and was also observed in four of fourteen YSTs affecting children less than 5 years old. The YSTs showed an increased frequency of 1p loss (p = 0.003), 3p gain (p = 0.02), 4q loss (p = 0.07) and 6q loss (p = 0.004) compared to the germinomatous tumors [30].

Among these miRNAs, miR-142-5p and miR-146a are upregulated in the pediatric germinomas (GP group) when compared to the NGMGCTs (IPG/PPG). Up to the present, no miRNA profile of pediatric GCTs has been published. A miRNome report on adult gonadal GCTs showed that, for each GCT subtype, the miRNA patterns are quite different [24]. In their dataset, miR-142-5p and miR-146a are also more abundant in adult seminomas than in gonadal ECs [24]. In addition, let-7e, miR-133b, miR-218 and miR-654-3p are also abundant in both pediatric NGMGCTs and adult ECs (Figure ​(Figure1C)1C) [24]. However, the notable discrepancies are miR-181c and miR-218, the expression levels of which are more abundant in adult testicular seminomas but are lower in pediatric intracranial germinomas (Figure ​(Figure1)1) [24]. The unique expression pattern of these miRNAs in pediatric CNS GCTs may reflect the differences in pathogenesis mechanisms between adult and pediatric GCTs [17], or, alternatively, the variation in genetic makeup between Western and Taiwanese patients.

We also correlated the transcript levels of miRNAs to their candidate targets in order to identify microRNA-mRNA target pairs (Table ​(Table3).3). It has been shown that some miRNAs, such as miR-1, can downregulate the transcript levels of a large number of target genes in mammalian cells [18]. Two large scale proteomic studies published very recently have shown that, although some microRNA target proteins are repressed without detectable changes in mRNA levels, more than a third of translational repressed targets also display detectable mRNA destabilization and, for the more highly repressed targets, mRNA destabilization usually makes up the major component of repression [19,20]. Gene expression microarrays can therefore be, and have been, applied for the identification of downstream targets for miRNAs [44-46]. However, proof of direct binding between those miRNAs and target mRNAs, as well as the direct translational repression of target mRNAs, is still needed. Such confirmation will require more wetlab experiments such as immunoblotting and reporter assays.

When compared with NGMGCTs, the germinomas largely recapitulate the features of self-renewing pluripotent human embryonic stem (hES) cells, such as involvement of POU5F1 (OCT4), NANOG and KLF4 (q < 0.01). Both seminomas and embryonal carcinomas are known to express stem cell markers, such as POU5F1 and NANOG [47,48]. In addition, in an attempt to find coordinated overexpressed gene clusters in GCTs, Korkola et al. found NANOG at chromosome 12p13.31 is overexpressed in undifferentiated (embryonal carcinomas and seminomas) tumors versus differentiated (teratoma, yolk sac tumor, and choriocarcinoma) tumors [16]. By overexpressing POU5F1, NANOG and KLF4, it is now possible to reprogram the transcriptomes of somatic primary cells, which results in their dedifferentiation from matured cells to ES cell-like iPS (induced pluripotent stem) cells [49]. The abundant expression of these dedifferentiation factors in germinomas therefore mirrors the more undifferentiated histopathological characteristics of these tumors. Whereas such similarities have previously been described for adult and pediatric seminomas [16,17,47,48], we now know that this also applies to Asian pediatric CNS germinomas.

Although germinomas abundantly express the above three stemness factors, it is NGMGCTs (IPG/PPG) who show a closer gene expression pattern to ESCs (Figure ​(Figure2C).2C). This observation is consistent with pervious global gene expression reports whereby the gene expression patterns of human ES cell lines are similar to those of the human embryonal carcinoma cell samples but are more distantly related to those of seminoma samples [12]. The close relationship between NGMGCTs and ES cells supports the hypothesis that germinomas are closely related to primordial germ cells (PGCs), and EC cells/NGMGCTs represent a reversion to a more ICM- or primitive ectoderm-like cell type [12]. Whether germinomas and zygotes/blastomeres share similar mRNA or microRNA profiles is under investigation at present. The close relationship between NGMGCTs and ES cells may additionally be reflected in the worse prognosis for these tumors. Recently, via novel genomic approaches, it has been shown that aggressive and poor prognostic tumors, such as glioblastomas, inherit preferential ES cell gene expression profiles [36]. The similarity between pediatric NGMGCTs and human ES cells may therefore reflect the clinical observation that CNS NGMGCTs are more malignant and show a higher fatality rate than germinomas.

The close relationship in genetic makeup between NGMGCTs and ESCs also suggest that factors other than POU5F1 (OCT4), NANOG or KLF4 are responsible for ESC gene expression. In this study, we found that two key epithelial-mesenchymal transition (EMT) regulators, SNAI2 (SLUG) and TWIST2, are abundantly expressed in the NGMGCT group (IPG/PPG) (Table ​(Table22 and Figure ​Figure2D).2D). It has been reported that EMT transcription factors, SNAI1 (alias SNAIL) and TWIST, can independently dedifferentiate mammalian cancer cells and induces the generation of cancer stem-like cells, which then form mammospheres [50]. It is possible that SNAI2 (SLUG) and TWIST2 behaves like Snail and TWIST and can introduce malignancy and stemness in pediatric GCTs. Targeting oncogenic stemness genes or EMT-related embryonic signaling pathways (such as the Wnt pathway, Figures ​Figures2D2D &3C) may differentiate a highly malignant NGMGCT into a more matured transcriptome type, thereby increasing the sensitivity of these tumors to the classical therapeutic regimen of radical resection, irradiation and chemotherapy, which would produce a better prognosis for the patients.

In addition to stemness genes (such as genes involved in reproduction and male gonad development), the germinomas were found to overexpress genes involved in the DNA damage checkpoint, which indicates active DNA integrity checking in the germinomas and thereby reflects why the clinical phenotype of germinomas has a better prognosis (Figure ​(Figure3A).3A). Among the other genes that were found to be expressed abundantly in germinomatous tissues were genes associated with the immune system process and this correlates with the abundant lymphocytic infiltration of germinomas found during histological observation. Relative to germinomas, we observed a significant enrichment of overexpression of differentiation and morphogenesis (especially neurogenesis) genes in NGMGCTs, which correlates with the differentiated state of these tumor cells (Figure ​(Figure3B).3B). There is also evidence of overexpression of genes in the Wnt/β-catenin pathway in our dataset (Figures 3B-C), which is consistent with previous studies of nonseminomatous malignant GCTs [15,51]. In concordance with the higher recurrence and disseminating clinical behaviors of NGMGCTs, a significant enrichment for overexpression of motility, tight junction, focal adhesion, and adherent junction genes in NGMGCTs was observed (Figures 3B-C). Our results thereby integrate molecular profiles with clinical observations and provide a better understanding of the underlying molecular mechanisms. The combined targeting of hub genes involved in all these biological modules by a cocktail therapy-like regimen may eventually lead to an alleviation of these malignant CNS tumors.

During the submission of this manuscript, a very recent reference based on testis GCTs identified gene expression signatures that predicted outcomes in patients with extra-cranial adult GCTs [52]. We compared the age and tumor characteristics between our series against the genomic study group of CNS GCTs in children and the reported study of extra CNS GCTs in adult men (Additional file 1-C) [52]. In our series and the genomic study of CNS GCTs, both germinomas and NGGCTs in children younger than 18 years old were included, whereas Korkola's study involved adult men with nonseminomatous GCTs (NSGCTs) [52]. In our series, 118 tumors were pure germinomas or tumors with a germinoma component, 49 tumors were pure teratoma or tumors with a teratoma component, and 27 cases were classified as YSTs including 10 pure YSTs, 11 tumors with a YST component, and 6 cases with serum AFP elevation (pure immature teratomas excluded). Among the 21 cases with genomic studies, 9 tumors were pure germinomas, 2 tumors were pure mature teratomas, and 9 tumors were mixed GCTs, including one mature teratoma with serum AFP elevation and one germinoma with serum AFP elevation. The correlation of tumor characteristics between the studies of Korkola et al. and ours in Additional file 1-C constituted the basis for the comparison of genomic molecular findings across the different therapeutic prognostic groups and histology between these two studies.

Korkola et al. concluded that using a 140-gene signature, they could predict 5-year overall survival (OS) (p < 0.001) [52]. Both our study and that of Korkola et al. identified good outcome GCTs express gene sets involved in immune function and the repression of differentiation (such as POU5F1/OCT4), while poor outcome GCTs express genes involved in active differentiation (in particular, neuron differentiation) (Fig. ​(Fig.3)3) [52]. A 10-gene prognosis model was also built using a univariate Cox model. When the samples were dichotomized by median score, there was significant separation of the survival curves (p < 0.002) [52]. These 10 genes were STX6, CFLAR, FNBP1, ITSN2, SYNE1, MAP3K5, PTGDS, PXMP2, IRAK4, and RABGAP1L [52]. Among these 10 genes STX6 (syntaxin 6) and CFLAR (CASP8 and FADD-like apoptosis regulator) are over-expressed in our germinoma group (q < 0.01). It will be interesting to fit their prognosis signatures onto our dataset to see whether GCTs of different anatomic locations, ages and ethnic populations express similar prognosis genes. However, since all the tissues used in our study were freshly collected over the last 2 years, only one death has been recorded so far (Additional file 1). As a result, this work needs to be carried out at a later stage.

The variation in chromosome copy number variation (CNV) regions between germinomas and NGMGCTs were mapped to cytobands 4q13.3-4q28.3 and 9p11.2-9q13 (Figure ​(Figure4).4). Chromosome abnormality analysis of adult testicular germ cell tumors (tGCTs) revealed that all GCTs show 12p gain [25,26]. In 2007, Palmer et al. used metaphase-based comparative genomic hybridization (CGH) to analyze genomic imbalance in 34 pediatric GCTs (22 yolk sac tumors (YSTs), 11 germinomatous tumors and one metastatic embryonal carcinoma). The YSTs showed an increased frequency of 1p loss (p = 0.003), 3p gain (p = 0.02), 4q loss (p = 0.07) and 6q loss (p = 0.004) compared to germinomas [30]. Most of their cases were from the testis, the ovary or the sacrococcygeal region and only 2 germinomas and 1 YST brain GCTs were included [30]; this is a possible explanation of the discrepancies between their results and ours. We also observed 4q loss in the NGMGCTs (including YSTs), suggesting that genomic imbalance in this region, and the genes/miRNAs encoded by this chromosomal region, may play a crucial tumor suppressing role during NGMGCT pathogenesis and affect clinical performance (Table ​(Table4).4). Six genes (BANK1, CXCL9, CXCL11, DDIT4L, ELOVL6 and HERC5) within 4q13.3-4q28.3 showed higher expression levels in the germinomas (Table ​(Table4).4). DDIT4L, ELOVL6 and HERC5 are among the top 50 highly expressed genes in germinomas (Table ​(Table1).1). A putative GCT tumor suppressor gene SYNPO2 (Synaptopodin 2), also known as myopodin, is also within the 4q13.3-4q28.3 deletion region (Table ​(Table4).4). SYNPO2 has recently been shown to have the highest predictive value when assessing 5-year overall survival [52], which is consistent with a possible role as a tumor suppressor. However, we do not observe differential SYNPO2 expression between NGMGCTs and germinomas (Table ​(Table4).4). It is unclear whether SYNPO2 expression is also downregulated in Taiwanese germinomas compared to normal brains. In addition, whether survival predictors derived from Western cases can be applied to Asian patients still awaits elucidation.

Recently two independent genome-wide association studies (GWAS) have reported on susceptibility loci associated with tGCT: Kanetsky et al. mapped seven markers at 12p22 near KITLG (c-KIT ligand) and two markers at 5q31.3 near SPRY4 (sprouty 4) [53]; furthermore Rapley et al. identified loci on chromosome 5, 6 and 12 [54]. A third locus, in an intron of BAK1, a gene that promotes apoptosis, was also identified by Rapley et al. [54]. Similarly, the CGH profiles in childhood GCTs have been reported to resemble those in adults [55,56]. In terms of cytogenetics differences between the different histological entities, loss of chromosome 19 and 22 material and gain of 5q14-q23, 6q21-q24 and 13q material were found to occur at a significantly lower frequency in seminoma adult tGCTs compared to non-seminoma adult tGCTs [25]. Among Taiwanese pediatric GCTs, no common copy number variation (CNV) could be found in either the germinomas or the mature teratomas (Figure ​(Figure4).4). The divergence between our results and published Caucasian ones may be partly due to the different ethnic samples used, the application of different bioinformatics algorithms and the fact that we compared the differences between germinomas and NGMGCTs but not common aberrations across all GCTs.